The present exemplary embodiment relates to the use of absorption detection strategies in obtaining information relating to a polymerase chain reaction (PCR) assay. The present exemplary embodiment also relates to the use of light emitting diodes (LED's) or laser diodes, and particularly light sources such as these that emit light in the ultra-violet range, in spectrophotometers and for detecting polymerase chain reaction (PCR) products. The exemplary embodiment finds particular application in conjunction with real-time PCR assays, and will be described with particular reference thereto. However, it is to be appreciated that the present exemplary embodiment is also amenable to other like applications.
The polymerase chain reaction (PCR) is a powerful technique, which results in the rapid production of multiple copies of a target nucleic acid sequence. The PCR technique has made it possible to analyze DNA fragments in samples that contain amounts of DNA that are either too small, or too degraded, to permit other types of nucleic acid analysis.
The PCR method is a cycling reaction in which template DNA is denatured by heating to separate the strands of the molecule. Primer (20–30 base fragments of DNA complementary to a region of the template) is annealed to the single-stranded templates. The cycle ends as the primer molecules are elongated by the action of DNA polymerase to produce molecules that are identical copies of the original template. Because the products of one PCR cycle can act as templates for the next PCR cycle, the number of new identical molecules produced doubles with each repetition of the cycle.
PCR is an immensely valuable technique which is very widely practiced, and has revolutionized the field of molecular biology. The technique is disclosed in detail in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188, all of which are hereby incorporated by reference. The reaction is typically carried out in solution in small reaction vessels, where the DNA to be amplified is in suspension. Apparatuses for this process are disclosed in U.S. Pat. No. 5,038,852 and in U.S. Pat. No. 5,475,610, both of which are hereby incorporated by reference. Additional PCR references include “PCR Protocols: A Guide to Methods and Applications,” M. A. Innis, et al., eds., Academic Press, New York, pp. 272–281; and Micklos, D. A. and G. A. Freyer, 1990, “DNA Science: A First Course in Recombinant DNA Technology,” Carolina Biological Supply Company, Burlington, N.C. and Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., all of which are hereby incorporated by reference.
PCR technology is unique in its ability to locate and exponentially amplify a small quantity of a specific nucleotide sequence which is “lost” against a large background of total nucleic acid. This feature of PCR has made possible the development of a vast number of experimental and diagnostic molecular biology techniques, which were previously extremely time consuming or, in many cases, impossible to perform.
Nucleic acids in a sample are usually first amplified by the PCR method and subsequently detected. This sequential approach is based on a single end-point measurement after the PCR is completed. The amount of amplified product observed at the end of the reaction is very sensitive to slight variations in reaction components because PCR is typically exponential. Therefore, the accuracy and precision of quantitative analysis using endpoint measurements is poor. Furthermore, endpoint measurements can produce a hook effect whereby high concentrations of a target polynucleotide to be amplified yield inaccurately low values.
In contrast to end-point determinations of amplified polynucleotides, real-time monitoring of PCR product generation offers the possibility of better precision and accuracy in quantitative measurements because the measurements are taken during the exponential phase of the PCR process. In contrast to classical end-point measurements, multiple measurements are taken during real-time monitoring. During the exponential phase of the PCR process, none of the reaction components are limiting, and therefore the effects on accuracy of reaching a maximum signal are eliminated. Real-time monitoring of PCR is based on kinetic measurements offering a better and a more complete picture of the PCR process. A number of real-time monitoring methods have been developed, however the methods use fluorescent signals in all cases.
Traditional PCR uses a thermostable DNA polymerase in a DNA synthesis reaction, primed by DNA oligonucleotides that are complementary to a specific sequence within the target DNA. Standard PCR (in the absence of probe DNA) results in a doubling of the number of copies of target sequence after each round of DNA synthesis, and a geometric increase in the number of copies after each reaction cycle. The product can be observed afterwards by separation of the DNA by agarose gel electrophoresis.
Real-time fluorescent PCR works similarly, with the addition of a third small fragment of DNA to the reaction mixture. The DNA/RNA detection reaction combines standard PCR with a third reagent, a probe DNA molecule that hybridizes to a target sequence between the sequences bound by the two PCR primers. The probe is labeled at one end with a fluorescent dye molecule and at the other end with a molecule that quenches the fluorescence of the dye molecule, such that the proximity of these two molecules results in a quenching of the dye's fluorescence. When a DNA polymerase extends one of the two primers, the probe molecule degrades and releases the fluorescent and quencher molecules bound to the ends of the probe. The separation of the dye and the quencher results in an increase in the overall fluorescence of the sample mixture. A detector in the PCR instrument continually monitors and records the fluorescence present in the sample. Significant accumulation of fluorescence in the sample above background level indicates a positive detection of the target DNA. Additional information concerning fluorescent PCR includes “Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions,” Biotechnology (New York), 1993, September. 11(9): 1026–30; “Simultaneous Amplification and Detection of Specific DNA Sequences,” Biotechnology (New York) 1992 April, 10(4): 413–7; “Real-time Quantitative Polymerase Chain Reaction: A Potential Tool for Genetic Analysis in Neuropathology,” Brain Pathol. 2002 January, 12(1): 54–66, Review; “Sensitivity of Multiplex Real-time PCR Reactions, Using the LightCycler and the ABI PRISM 7700 Sequence Detection System, Is Dependent on the Concentration of the DNA Polymerase,” Mol Cell Probes, 2002 October, 16(5): 351–7; “Real Time Quantitative PCR,” Genome Res. 1996 October, 6(10):986–94, all hereby incorporated by reference.
Since its inception a few years ago, real-time quantitative PCR-based assays have become an indispensable part of biological research. The fields of biotechnology and biopharmacology rely on this technology for high throughput screening of plausible genetic loci. This assay incorporates, and thus is limited by, the use of labeling fluorochromes, fluorescent primers, and Taqman probes. These fluorochromes or dye markers are expensive and their detection requires large integrated light sources and detection optics on the system. For example, an integrated real-time PCR system can cost $90,000 (Applied Biosystems ABI PRISM® 7700 Sequence Detection System) as compared to $7,500 for a non-real time PCR system (GeneAmp 9700). Both systems perform PCR amplification for 96 wellplates but the GeneAmp 9700 requires a separate spectrophotometer or a fluorescent wellplate reader for DNA concentration measurement. Accordingly, there is a need to avoid the use of fluorochromes and associated detection light sources and optics otherwise required when conducting real-time PCR analysis.
The present exemplary embodiment contemplates a new and improved spectrophotometer and PCR system, and related methods, that utilize absorption detection strategies for detecting, analyzing, or quantifying PCR products, which overcome the above-referenced problems and others. The exemplary embodiment also contemplates LED's or lasers as light sources in such devices, particularly for conducting PCR-based assays. In a particular aspect, the light is in the ultra-violet wavelength range. Additionally, the present exemplary embodiment also contemplates a new and improved real-time PCR-based assay that avoids many of the problems associated with previously known systems and techniques.